Method for the preparation of a carminic acid lake

ABSTRACT

A method for the preparation of carminic acid lakes, novel carminic acid lake compositions and their uses in foods, such as yogurt, fruit preparations, beverages, other miscellaneous food products that may need a stable red color, and in cosmetics.

FIELD OF THE INVENTION

The present invention relates to a method for the preparation ofcarminic acid lakes, novel carminic acid lake compositions and theiruses in foods, such as yogurt fruit preparations, beverages, othermiscellaneous food products that may need a stable red color, and incosmetics.

BACKGROUND OF THE INVENTION

Carminic acid is a colorant, which can be extracted from the femaleinsect bodies of Dactylopius coccus costa (alternative name Coccus cactiL.). The insects live on Nopalea coccinellifera, Opuntia fidus indicaand other plants of the family Cactaceae cultivated for instance in thedesert areas of Mexico and Central and South America. Depending on thepH the colorant may be a color in a spectrum from orange over red topurple and is generally known as cochineal or cochineal color. Carminecolorant is widely used in foods and beverages.

Carminic acid is harvested by extraction from said insects' dried bodieswith water or alcohol. During the aqueous based extraction of carminicacid from the insect, an amount of insect protein is also released fromthe insect and will be contained in the color extract. The level ofinsect protein is typically less than 0.5%. The aqueous based extract ofcochineal is primarily containing carminic acid plus some cochinealprotein and other minor extractable substances from the insect.Hereinafter this extract is referred to as cochineal extract solution.

It has been reported that the cochineal insect proteins could createsome allergy related problems. In order to solve this allergy problem,US2002/0058016; EP1318178 (SAN-EI GEN (Japan)) describes a processwherein the cochineal extract solution is subjected to enzymaticproteolysis and Dactylopius coccus costa insect proteins with amolecular weight greater than 6,000 is subsequently removed.

The art describes numerous other not proteolysis based standard methodsto make a pure carminic acid product substantially free of Dactylopiuscoccus costa insect proteins. Such methods generally use suitableadsorption treatments, ion exchange treatments, acid treatments and/ormembrane treatments. Commercially available carminic acid productssubstantially free of Dactylopius coccus costa insect proteins include awater-soluble Cochineal powder from Chr. Hansen, A/S (Denmark) orsimilar commercially available products from e.g. the companies Pronex(Peru) or Sensient.

A carminic acid lake denotes herein a type of coloring compositionconsisting essentially of carminic acid combined more or less definitelywith aluminum and calcium. This is termed carminic acid calcium-aluminumlake. The lake is prepared by reacting carminic acid with aluminumand/or calcium under aqueous conditions. The conditions are adjusted ina way that favors precipitation of the aluminum/calcium-carminic acidlake complex composition. This complex composition is termed carmine.

The working example 2, US2002/0058016 (SAN-EI GEN (Japan) describespreparation of a carminic acid calcium-aluminum lake based on a purecarminic acid solution made in example 1. Example 2 section [0085] thenexplains that “when a carminic acid aluminum lake or calcium lake(Carmine) is caused to form a polymer, there is the tendency that when alow molecular protein is allowed to be present, a more neat lake can beobtained with an increased intensity of red color. Therefore, acochineal color (carmine) with a higher intensity of redness and anincrease market value may be prepared by adding a protein ofcomparatively low molecular weight, which does not become an allergen tothe above allergen-free solution.” Besides this speculative statement,no further relevant details with respect to a possible addition of lowmolecular weight protein to the carminic acid lake are described.

SUMMARY OF THE INVENTION

The aim of the present invention is to provide an improved carminic acidlake, which is free of potentially allergenic cochineal insect proteins.

The solution is based on several relevant parameters important for thepreparation of an optimal lake that has been identified by the presentinventors by starting from a pure carminic acid product substantiallyfree of Dactylopius coccus costa insect proteins. These parameters aredescribed in further details below and include details regarding addednon cochineal insect proteins and carminic acid percentage.

Accordingly, a first aspect of the invention relates to a process formaking a carminic acid lake comprising the steps of:

-   -   (i) treating an aqueous solution,        -   comprising        -   (a) 0.5 to 15% (g/l) carminic acid and        -   (b) from 0.75 to 15% (g/l) not Dactylopius coccus costa            insect proteins, where substantially all of the not            Dactylopius coccus costa insect proteins have a molecular            weight (MW) below 10,000 Da,        -   wherein the aqueous solution is substantially free of            Dactylopius coccus costa insect proteins with a molecular            weight (MW) above 6,000 Da;    -   with an aluminum and calcium compound,    -   (ii) adjusting the conditions to get suitable conditions to        produce a calcium-aluminum carminic acid lake, wherein the lake        comprises        -   (a) from 40 to 80% carminic acid (dry weight) and        -   (b) from 10 to 30% not Dactylopius coccus costa insect            proteins (dry weight), where substantially all of the not            Dactylopius coccus costa insect proteins have a molecular            weight (MW) below 10,000 Da and        -   wherein the lake is substantially free of Dactylopius coccus            costa insect proteins with a molecular weight (MW) above            6,000 Da.

A commercially produced cochineal extract solution normally comprisesaround 0.6% carminic acid and relatively low amounts Dactylopius coccuscosta insect proteins, generally around 0.1 to 0.25% of proteins. In theart this has been regarded as a sufficient amount of proteins to producea lake. Contrary to the art, the present inventors identified thatimproved lakes may be obtained by including higher amounts of proteinsas described herein. An advantage of using significantly higher amountsof proteins, as described in step (i) above, is that the yield ofcarminic acid is improved. The yield relates to the amount of carminicacid in the lake of step (ii) as compared to the initial amount in theaqueous solution of step (i). See working examples herein for furtherdetails, where yields up to 98.2% have been obtained.

At the filing date of the present application, the present inventorswere not aware of any prior art (for instance commercial lake products)that describes carminic acid lake with the amounts of carminic acid andnon insect proteins as described herein.

Accordingly, a second aspect relates to a carminic acid calcium-aluminumlake obtainable by a process of the first aspect and embodiments thereofas described herein, wherein the lake comprises

-   -   (a) from 40 to 80% of carminic acid (dry weight) and    -   (b) from 10 to 30% of not Dactylopius coccus costa insect        proteins (dry weight), where substantially all of the not        Dactylopius coccus costa insect proteins have a molecular weight        (MW) below 10,000 Da and    -   wherein the lake is substantially free of Dactylopius coccus        costa insect proteins with a molecular weight (MW) above 6,000        Da.

The word “obtainable” shall be understood in the normal English sense ofthe word, i.e. as the lake may be obtained by the specified process butcould in theory be obtained in a different manner. Preferably, the lakeof the second aspect is obtained by a process of the first aspect andembodiments thereof as described herein.

A third aspect of the invention relates to use of a carminic acidaluminum lake, a carminic acid calcium lake or a carminic acidcalcium-aluminum lake of the second aspect and embodiments thereof asdescribed herein for making a color composition to be added to foodproducts, sweets, confectionary, beverages, pharmaceuticals orcosmetics.

This third aspect may alternatively be described as a method forcolouring food products, sweets, confectionary, beverages,pharmaceuticals or cosmetics comprising use of a color composition madefrom a carminic acid aluminum lake, a carminic acid calcium lake or acarminic acid calcium-aluminum lake of the second aspect and embodimentsthereof as described herein.

DETAILED DESCRIPTION OF THE INVENTION

A Process for Making a Carminic Acid Lake

The carminic acid to be used to make the aqueous solution of step (i)shall preferably be from a pure carminic acid composition. A “purecarminic acid composition” denotes herein a highly purified cochinealextract solution. Preferably it comprises from 90 to 99% carminic acid(dry weight) and is preferably substantially free of Dactylopius coccuscosta insect proteins.

The term “substantially free of Dactylopius coccus costa insectproteins” denotes herein that it is not detectable by a SDS-PAGEanalysis. A suitable SDS-PAGE analysis is given in Example 1.

A pure carminic acid composition may be made in a number of routine wayse.g. based on methods that generally use suitable adsorption treatments,ion exchange treatments, acid treatments and/or membrane treatments.Commercially available pure carminic acid compositions include awater-soluble Cochineal powder from Chr. Hansen, A/S (Denmark) orsimilar commercially available products from e.g. the companies Pronex(Peru) or Sensient. A pure carminic acid composition may be made by theproteolysis-based method described in US2002/0058016.

The not Dactylopius coccus costa insect proteins used to make theaqueous solution of step (i) shall preferably be from a proteincomposition obtained from a source that is acceptable for the foodindustry and/or pharmaceutically acceptable. For example, the proteinsshould preferably not be based on a composition of proteins obtainedfrom e.g. a snake known to be toxic for e.g. humans. It shouldfurthermore preferably be from a source known to cause no or onlyacceptable allergy related problems.

The term “not Dactylopius coccus costa insect proteins” used to make theaqueous solution of step (i) globally denotes herein peptides, aminoacids, proteins or mixtures thereof.

Examples of suitable protein compositions are based on plant proteinssuch as soya proteins and potato proteins.

Preferably, the proteins are milk proteins such a cow milk proteins.Preferably, the milk proteins are obtained from a milk-based hydrolysatesuch as a whey hydrolysate. Suitable commercially available wheyhydrolysate may be obtained from commercial dairy companies. This isparticularly relevant when the carminic acid color is to be used in amilk based food product.

Preferably, substantially all of the not Dactylopius coccus costa insectproteins have a molecular weight (MW) below 6,000 Da, more preferablybelow 4,000 Da and even more preferably below 3,000 Da.

It is implicit in the process as described herein that when the proteinsof step (i) have a certain MW then the resulting lake of step (ii) willalso get a corresponding protein MW composition. For instance, ifsubstantially all of the proteins of step (i) have a molecular weight(MW) below 6,000 Da then substantially all of the proteins in the lake,of step (ii) will have a molecular weight (MW) below 6,000 Da.

The term “substantially all of the proteins have a molecular weight (MW)below [a number] Da” denotes herein that proteins with a greater MW thanindicated are not detectable by a SDS-PAGE analysis. A suitable SDS-PAGEanalysis is given in Example 1.

An aqueous solution may preferably be prepared simply by adding asuitable protein composition (e.g. in powder form) to a pure carminicacid solution or simply by adding e.g. pure carminic acid (e.g. inpowder form) to a solution comprising a suitable protein composition. Inother words, simply by mixing a suitable protein composition with asuitable pure carminic acid composition.

In the aqueous solution of step (i) of the first aspect it is preferredthat the ratio Carminic acid/not Dactylopius coccus costa insectproteins (g/l) is less than 1. The present inventors have identifiedthat if the ratio is greater than 1 the solution has a tendency to gel.See table of working example 1 herein.

Preferably, the ratio Carminic acid/not Dactylopius coccus costa insectproteins (g/l) is from 0.4 to 0.9, more preferably from 0.5 to 0.8 andmost preferably from 0.5 to 0.7.

Before the proteins are added to the aqueous solution, the pH of thesolution shall preferably be adjusted to a pH from 5 to 7, morepreferably around pH 6. Preferably, the aqueous media is water,preferably distilled water.

Preferably, the aqueous solution is substantially free of Dactylopiuscoccus costa insect proteins.

Preferably, the aqueous solution comprises from 1 to 10% carminic acidand from 1.5 to 10% not Dactylopius coccus costa insect proteins, morepreferably the aqueous solution comprises from 2 to 7% carminic acid andfrom 3 to 8% not Dactylopius coccus costa insect proteins, even morepreferably the aqueous solution comprises from 3 to 5% carminic acid andfrom 4 to 6% not Dactylopius coccus costa insect proteins.

The carminic acid/protein aqueous solution is treated with the aluminumand calcium compound under suitable conditions to produce the carminicacid lake. The skilled person generally knows suitable conditions tomake a lake. However, the present inventors have analyzed this in detailand the preferred conditions are explained below.

Treatment of the aqueous solution with an aluminum and calcium compoundmay be performed in different ways. It may e.g. be performed by addingthe aluminum and calcium to a solution only comprising the proteins andthen thereafter by adding the carminic acids to the solution.

However, it is preferred to first make an aqueous solution comprisingboth the carminic acid and the proteins and then add the aluminum andcalcium compound to this solution. Without being limited by theory, itis believed to be important for optimal lake formation that the aluminumand calcium are added to a solution already comprising both the carminicacid and the proteins.

In fact this is different to the suggestion in working example 2 ofUS2002/0058016, where a carminic acid aluminum solution is first madeand it is then suggested to add low MW proteins to this solution.

It is preferred to first add the aluminum and then wait for a suitabletime before the calcium is added to the aqueous solution. A suitabletime is around from 30 seconds to 1 hour, preferably from 1 minute to 15minutes.

Surprisingly, the present inventors have identified that if calcium isadded to the carminic acid/protein solution first, then there is a riskof unwanted “black spots” developing in the final lake. The black spotsare believed to be calcium carminate. There are created far less “blackspots” when aluminum is added first to the carminic acid/protein aqueoussolution. See working example 2 herein for further details.

Preferably, the aqueous solution is treated with from 1 g aluminum/lsolution to 20 g aluminum/l solution and from 1 g calcium/l solution to20 g calcium/l solution. The skilled person knows how to optimize thisin relation to the desired color of interest.

A suitable aluminum compound is aluminum sulphate. Preferably thealuminum compound is AlK(SO₄)₂*12 H₂O. A suitable calcium compound iscalcium carbonate or Calcium chloride. Preferably the calcium compoundis (CH3COO)₂Ca*H₂O.

During the treatment of the carminic acid/protein aqueous solution withthe aluminum and/or calcium compound it is preferred to adjust the pH toa value from 3 to 7 in order to produce the carminic acid lake.Preferably, the pH is adjusted to a value from 4 to 6 and morepreferably adjusted to a pH value from 4.5 to 5.5.

The adjustment of the pH may be done after the carminic acid/proteinshas been combined with the salts. However, preferably the adjustment isdone before addition of salts.

Preferably the carminic acid/protein aqueous solution is treated withthe aluminum and calcium compound under following suitable conditions toproduce the carminic acid lake:

-   -   time period: 15 minutes to 2 hours, more preferably from 30        minutes to 60 minutes    -   temperature: from 80° C. to 99° C., more preferably from 92° C.        to 97° C.

The final composition of the produced carminic acid lake of step (ii)relates to the initial composition of the aqueous solution of step (i).The preferred carminic acid/protein contents of the aqueous solution aregiven above. Corresponding preferred compositions of the producedcarminic acid lake of step (ii) are wherein the lake comprises from 55to 75% carminic acid (dry weight) and from 15 to 25% proteins (dryweight), more preferably wherein the lake comprises from 62 to 72%carminic acid (dry weight) and from 18 to 22% proteins (dry weight).

Of course the amount of carminic acid and proteins in the lake cannot bemore than 100%. Actually, the sum will generally be around 90% since thelake will comprise other material such as the salts used to produce thelake. Accordingly, if the lake comprises 75% carminic acid (dry weight)then it would normally not have more than 15% proteins (dry weight).

A preferred method to determine the amounts of carminic acid is a knownstandard method described by FAO/WHO: Document from 44^(th) session ofthe JECFA committee in 1995 and Food Chemical Codex, second edition(FCCII). In Commission Directive 94/45/EC from July 1995, Officialjournal of the European Communities No. L 226, p. 9, it is describedthat the proper diluted color product E-120 has a maximum at 494 nm.

A preferred method to determine the amount of proteins is the knownKjeldahl method and/or by amino acid analysis.

After the carminic acid lake of step (ii) is produced it is routine toinclude adequate subsequent steps. These may include a step of filteringthe carminic acid lake and/or a step of drying the carminic acid lake toget a dried carminic acid lake. These steps are routine for the skilledpersons and reference is made to the reference textbook given above.

Use of a Carminic Acid Lake in Different Products

In order to use the carminic acid lake, as described herein, it shouldbe converted into a suitable color composition. The term colorcomposition should be understood broadly. Depending on the final needsit may be the carminic acid lake as such or maybe the lake afterfiltering and/or drying. Alternatively, the lake may be converted into asuitable carmine solution or water-soluble carmine (powder).

However, it may also be a color composition where a suitable carrier oradditive which is food sanitation-wise or pharmaceutically acceptablehas been added. The specific choice of carrier or additive will dependon the final use, e.g. in food or pharmaceuticals, and the skilledperson may routinely select adequate carriers or additives. For furtherdetails, see US2002/0058016.

As said above, the color composition may be added to food products,sweets, confectionary, beverages, pharmaceuticals or cosmetics.

The term food product denotes herein both food and feed products.Preferably it is food products. Preferred food products include dairyproducts such as yogurt or meat.

A further relevant feature of the color composition is the strength ofthe color. The color of the carminic acid may e.g. be red and the colorcomposition may be made with different strength of e.g. red in order toget an adequate color in the final e.g. food product.

Starting from the carminic acid lake, as described herein, it is routinework to make adequate steps to get a color composition with requiredcolor strength.

The color composition may be encapsulated, using conventional microencapsulation techniques suitable for food products, for example, asdescribed in WO97/26803 (Chr. Hansen A/S), by encapsulation in gumarabic.

EXAMPLES Materials and Analytical Methods

Pure carminic acid: Commercially available carminic Acid obtained frompurified cochineal extract (Dactylopius coccus costa). Minimum around90% Carminic Acid.

Protein composition: Commercial whey protein hydrolysate suitable asprotein source in infant formulas with reduced allergen content.Substantially all of the proteins have a molecular weight (MW) below6,000 Da.

-   ELISA analysis: Antigenecity tested by one site immunometic assay    ELISA with polyclonal antibodies raised on whey protein concentrate-   μg protein eqv./g protein max 85-   Aluminum salt: AlK(SO₄)₂*12H₂O-   Calcium salt: (CH₃COO)₂Ca*H₂O-   Acetic Acid: CH₃COOH-   Sodium Hydroxide: NaOH-pellets    SDS-PAGE Analysis

Solvent Tris buffer pH 8 (20 g Tris/1,000 ml demi water, pH adjustedwith conc. HCl)

The lakes of carmine were added buffer solution so that all samplescontained the same amount Carminic Acid/ml corresponding to 1.5 g/l of a46.56% C.A. lake.

-   Marker 12 or Sea Blue is applied as the protein size indicator (MW)-   1M DL-Dithiothreitol (DTT): 20 mg DTT solution in 130 μl MQW-   Volume added to the gel: Mark 12 5 μl, Sea Blue 7 μl, Carmine    samples 10 μl-   NUPAGE Novex high performance pre-cast gel 4-12% BIS-TRIS are    stained by cromassie blue and/or silver

Example 1 Production of Carminic Acid Lake

This example discloses examples of preparation of Al—Ca carmine lakewith carminic acid and whey hydrolysate.

An amount of 21 g Carminic acid was dissolved in 500 ml water and inthis carmine solution 33 g peptide was dissolved. pH was adjusted to 5and the temperature was raised to 85-95° C. Then 8 g AlK(SO₄)₂*12H₂O wasdissolved in 50 ml water and added to the solution and then 8.2 g(CH₃COO)₂Ca*H₂O was dissolved in 50 ml water and added to the solution.After 45 minutes 70-98% of the carminic acid in the solution wasconverted to carmine lake, which precipitated from the solution. Thecarmine lake was separated from the solution, dried and analyzed.

% % C.A./ % Al % Ca % C.A. Protein Protein salt salt Yield Processing4.5 1.5 2.61 1.6 1.6 — Impossible 4.5 2.2 1.78 1.6 1.6 — Impossible 2.51.0 2.17 1.6 1.6 — Impossible 3.25 5 0.7 1.6 1.6 98.4 Easy 2 3 0.7 0.90.8 93.9 Easy 0.75 1 0.8 0.24 0.25 85.2 Easy 4 6.7 0.6 1.6 1.6 98.2 Easy4 5.7 0.7 1.6 1.6 97.7 Easy 4 5.0 0.8 1.6 1.6 97.4 Easy 4 4.4 0.9 1.61.6 95.0 Possible

The resulting carmine lakes had a carminic acid content of 65-75% and aprotein content of 18-22%. None of the proteins in the lakes, detectablein the SDS-PAGE analysis, were bigger than 3,000 Da.

The resulting carmine lakes have bright red color shades and wereapplicable as cochineal protein based carmine lakes for making carminesolutions and water-soluble carmine powders.

Conclusions

The results shown in the table in example 1 demonstrates the following:

-   (1) that optimal carminic acid yields are obtained when the aqueous    solution comprises from 3% to 5% carminic acid and from 4% to 7% of    whey protein hydrolysate;-   (2) when the amount of protein is increased, within the ranges as    described herein, the yield is improved;-   (3) that the ratio carminic acid/added proteins shall be less than 1    to avoid gelling and thereby impossible processing. Furthermore, the    preferred ratio is from 0.5 to 0.8 and most preferably around 0.6.

Example 2

This example discloses examples of Al—Ca carmine lake preparation withcarminic acid and whey hydrolysate.

An amount of 21 g Carminic acid was dissolved in 500 ml water and inthis carmine solution 33 g peptide was dissolved. pH was adjusted to 5and the temperature was raised to 85-95° C. Then 8 g (CH₃COO)₂Ca*H₂O wasdissolved in 50 ml water and added to the solution and then 8.2 gAlK(SO₄)₂*12H₂O was dissolved in 50 ml water and added to the solution.After 45 minutes 70-98% of the carminic acid in the solution had beenconverted to carmine lake, which precipitated from the solution. Thecarmine lake was separated from the solution, dried and analyzed.

Visible black spots in the lake were identified. The black spots weremost likely calcium carminate, which had not undergone the change fromcalcium carminate to aluminum-calcium carmine lake.

By adding the AlK(SO₄)₂*12H₂O solution first, it is believed, that thealuminum and peptide attach to the carminic acid and makes itdifficult/impossible for the calcium to form calcium carminate before itforms aluminum-calcium carmine lake.

Conclusions

The result in example 2 demonstrates that it is important to first addthe aluminum, wait for a period and then add the calcium in order to getless “black spots”.

1. A process for making a carminic acid lake, comprising treating an aqueous solution with an aluminium and calcium compound, said aqueous solution comprising from 0.5% to 15% of carminic acid and from 0.75% to 15% of protein that is milk-based hydrolysate, wherein (i) substantially all of said protein has a molecular weight below 10,000 Da and (ii) said aqueous solution is substantially free of Dactylopius coccus costa insect proteins with a molecular weight above 6,000 Da, under conditions such that a carminic acid lake is produced that comprises from 40% to 80% by dry weight of carminic acid and from 10% to 30% by dry weight of said protein, wherein substantially all protein of said carminic acid lake (i) has a molecular weight below 10,000 Da and (ii) is substantially free of Dactylopius coccus costa insect proteins with a molecular weight above 6,000 Da.
 2. The process of claim 1, wherein said milk-based hydrolysate comprises whey hydrolysate. 